Genetic insulation of the synthetic pathway. A) E. coli BL21(DE3) cysI cells were transformed with plasmids expressing zm FNR, so FD and zm SIR. The cysI deletion conveys a requirement for reduced sulfur, which the heterologous pathway supplies. Cells were grown for 24 hours on selective media with or without atmospheric O2. All three factors were required to rescue growth under aerobic conditions (left plate). Expression of zm FNR was not required under anaerobic conditions (right plate), indicating that so FD was receiving electrons from another source. B) Genetic deletions were targeted to eliminate potential endogenous redox partners for ferredoxin, therefore linking sulfide production specifically to a synthetic electron source. Also deleted were the catalytic subunits of each native hydrogenase, ensuring that only exogenous H2 was present in our system. C) Each deletion strain was transformed with so FD, zm SIR and either zm FNR or an empty plasmid. Growth was measured after 18 hours at 37°C under strict anaerobic conditions, as described in the methods. Sequential deletions reduced the nonspecific anaerobic background growth, with the largest effect produced by the deletion of ydbK. The final deletion strain, EW11, showed no growth defect in rich media and was used in all later experiments.